Molecular Diagnosis of Analbuminemia: A New Case Caused by a Nonsense Mutation in the Albumin Gene

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23–c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer

Structure and Properties of the C-terminal Domain of Insulin-like Growth Factor-binding Protein-1 Isolated from Human Amniotic Fluid

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules

Congenital analbuminemia caused by a novel aberrant splicing in the albumin gene

Introduction:Congenital analbuminemia is a rare autosomal recessive disorder manifested by the presence of a very low amount of circulating serum albumin. It is an allelic heterogeneous defect, caused by variety of mutations within the albumin gene in homozygous or compound heterozygous state. Herein we report the clinical and molecular characterization of a new case of congenital analbuminemia diagnosed in a female new-born of consanguineous (first degree cousins) parents from Ankara, Turkey, who presented with a low albumin concentration (< 8 g/L) and severe clinical symptoms. Materials and methods: The albumin gene of the index case was screened by single-strand conformation polymorphism, heteroduplex analysis, and direct DNA sequencing. The effect of the splicing mutation was evaluated by examining the cDNA obtained by reverse transcriptase - polymerase chain reaction (RT-PCR) from the albumin mRNA extracted from proband’s leukocytes. Results: DNA sequencing revealed that the proband is homozygous, and both parents are heterozygous, for a novel G>A transition at position c.1652+1, the first base of intron 12, which inactivates the strongly conserved GT dinucleotide at the 5’ splice site consensus sequence of this intron. The splicing defect results in the complete skipping of the preceding exon (exon 12) and in a frame-shift within exon 13 with a premature stop codon after the translation of three mutant amino acid residues. Conclusions: Our results confirm the clinical diagnosis of congenital analbuminemia in the proband and the inheritance of the trait and contribute to shed light on the molecular genetics of analbuminemia

Clinical, Genetic, and Protein Structural Aspects of Familial Dysalbuminemic Hyperthyroxinemia and Hypertriiodothyroninemia

Familial dysalbuminemic hyperthyroxinemia (FDH-T4) and hypertriiodothyroninemia (FDH-T3) are dominantly inherited syndromes characterized by a high concentration of thyroid hormone in the blood stream. The syndromes do not cause disease, because the concentration of free hormone is normal, but affected individuals are at risk of erroneous treatment. FDH-T4 is the most common cause of euthyroid hyperthyroxinemia in Caucasian populations in which its prevalence is about 1 in 10,000 individuals, but the prevalence can be much higher in some ethnic groups. The condition is caused by a genetic variant of human serum albumin (HSA); Arg218 is mutated to histidine, proline, or serine or Arg222 is changed to isoleucine. The disorder is characterized by greater elevation in serum l-thyroxine (T4) than in serum triiodothyronine (T3); T4 can be increased by a factor 8–15. The high serum concentration of T4 is due to modification of a binding site located in the N-terminal half of HSA (in subdomain IIA). Thus, mutating Arg218 or Arg222 for a smaller amino acid reduces the steric restrictions in the site and creates a high-affinity binding site. The mutations can also affect binding of other ligands and can perhaps cause modified pharmacokinetics of albumin-binding drugs. In normal HSA, the high-affinity site has another location (in subdomain IIIB). Different locations of these sites imply that persons with and without FDH-T4 can have different types of interactions, and thereby complications, when given albumin-binding drugs. FDH-T3 is caused by a leucine to proline mutation in position 66 of HSA, which results in a large increment of the binding affinity for T3 but not for T4. For avoiding unwanted treatment of euthyroid persons with hyperthyroxinemia or hypertriiodothyroninemia, protein sequencing and/or sequencing of the albumin gene should be performed

The Structural characterization and bilirubin-binding properties of albumin Herborn, a [Lys240->Glu] albumin mutant

We report the molecular defect of albumin Herborn, a new genetic variant of human serum albumin which has been found in Germany. Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr 3 (residues 124–298). This fragment was isolated on a preparative scale and subjected to tryptic and V8 protease digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution Lys240→Glu. The -2 charge change of albumin Herborn, which is probably due to a A→G transition in the first position of the corresponding codon in the structural gene, has no significant effect on its electrophoretic mobility under non-denaturating conditions. Therefore we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 453–459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The apparent equilibrium association constants (Ka±SD) and the number of high-affinity binding sites (n) of the defatted variant for bilirubin and biliverdin were Ka= 1.03 ± 0.18 × 108 M−1, n= 1.07; and Ka= 7.48 ± 1.10 × 106 M−1, n= 1.01, respectively. The Ka values are about 93.3% and 99.1% of the values found for the normal protein under the same conditions. These results strongly suggest that Lys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high-affinity site